Several important results have changed our view of what processes are primarily responsible for point mutations. Heretofore, we imagined that most mutations might be caused by environmental mutagens. New evidence suggests a much greater role for spontaneous mechanisms. First we discovered that the specific G to A transition of the 12th codon in 85% of methyl- nitrosourea induced rat breast cancers was not induced by the chemical but arose in untreated animals in what appears to be a spontaneous burst of mutation at or near first estrus. Secondly a technologic advance within the Project has allowed us to directly observe mutational spectra in the DNA of human tissues. In a comparison of the spectra in a mitochondrial sequence, we find the sets of mutations arising in the TK-6 human cell line under pristine laboratory conditions are essentially the same as seen in colonic epithelial cells and skeletal muscle tissue obtained as surgical samples. Finally, research of this Project has shown us that the set of point mutational hotspots studied by our approach reveals not a small fraction of all mutants (as has been sometimes argued), but a set comprising more than 50% and perhaps more than 90% of the point mutations arising in the hprt gene of a human cell line. We now propose to transfer the mutational spectrometry technology throughout the Program so that it may also be used to study the biochemical genetics of spontaneous mutation in yeasts and bacteria. We further propose to extend the technology to measure mutational spectra in nuclear single-copy human genes. Emphasis is placed on cancer "gatekeeper" genes. We propose to continue our studies of spontaneous mutational spectra in human cells to determine the effects of mutations in DNA replication and repair genes.